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News Article | September 2, 2016

Cancer thrives when mutated cells undergo frequent division. Most anti-cancer drugs work by inserting themselves in between the DNA base pairs that encode our genetic information. This process is known as intercalation, and it can result in subtle changes to the DNA molecule’s geometric shape or tertiary structure. These structural changes interfere with the DNA’s transcription and a cell’s replication process, ultimately resulting in cell death. While intercalating agents used in chemotherapy drugs are highly effective in fighting cancer, they also may kill important cells in the body and lead to other complications such as heart failure. Therefore, researchers are always searching for faster, cheaper and more accurate tools to aid in the design of next-generation anti-cancer drugs with reduced side effects. A paper published in ACS Nano, one of the top nanotechnology journals in the world, explores this topic. “Modeling and Analysis of Intercalant Effects on Circular DNA Conformation,” focuses on the effect of the intercalating agent ethidium bromide (a mimic for many chemotherapy drugs) on the tertiary structure of DNA. Lead researchers on the project were Daniel Fologea, assistant professor in the Department of Physics, and David Estrada, assistant professor and graduate program coordinator, Micron School of Materials Science and Engineering. “The second dogma of biology states that structure determines function. Any structural change may be translated into a change of function,” says Fologea. “So we devised a simple method to allow us to ‘see’ how an intercalating agent is changing the shape of DNA at the single molecule level.” To achieve this, the team created a nanopore — a nanoscale sized opening in an ultrathin membrane — through which a single DNA molecule can pass when an electric field is applied to the microfluidics containing the device. When the DNA goes through this nanoscale aperture, it generates an electrical signal that provides information about its physical properties such as its shape, elasticity and even interactions with other biomolecules. “Our measurements revealed unique current blockades correlated to branched structures in the DNA molecule that resulted from ethidium bromide intercalation,” says lead author Eric Krueger, who was a postdoctoral researcher working jointly in Estrada and Fologea’s laboratories. “These results show that nanopores can be a scalable, efficient and cost effective way to measure DNA interactions with emerging anti-cancer drugs.” The team involved collaborations with the University of Illinois at Chicago and the University of Illinois at Urbana-Champaign. Using a combination of nanofabrication techniques, nanopore electrical measurements, atomic force microscopy imaging and all-atom simulations carried out on XSEDE supercomputer resources, the team’s interdisciplinary approach has extended the practical use of solid-state nanopores to a new area of cancer research. XSEDE (Extreme Science and Engineering Discovery Environment) provides researchers with access to several advanced computing resources across the country. “The new simulation capabilities developed by Dr. Khalili-Araghi’s group at the University of Illinois at Chicago have opened up a new computational space for the rapid screening of intercalating molecules as potential cancer therapies,” says Estrada. “We are now working on coupling such simulations with atomically thin materials, such as graphene and two-dimensional molybdenum disulfide, in order to increase the sensitivity of our approach.” This project highlights the capabilities of two of Boise State University’s newest Ph.D. programs, in biomolecular sciences and materials science and engineering, that have helped the university become designated as a doctoral research university by the Carnegie Classification of Institutions of Higher Education, the nation’s premier college classification system. The research was supported by the Division of Research and Economic Development and the Biomolecular Research Center at Boise State University.

News Article | November 16, 2016

A synthetic version of a rare toxin produced by a sea creature appears to hold promise for treating many different types of cancer while minimizing the harmful side effects of widely used chemotherapy drugs. A study published today in the journal Science Translational Medicine describes research on a substance called diazonamide, which prevents cell division, and was isolated from a marine animal called Diazona angulata. Led by Patrick Harran, UCLA's Donald J. and Jane M. Cram Professor of Organic Chemistry, researchers produced a synthetic form of diazonamide that, in rodents, appears to be effective in fighting breast cancer, colon cancer and leukemia. The compound the scientists synthesized, DZ-2384, is more potent and lasts longer in the bloodstream than natural diazonamide. And, when combined with a chemotherapy drug called gemcitabine (which is marketed under the brand name Gemzar), the compound is effective in rodents for combating advanced pancreatic cancer. DZ-2384 works by disrupting a molecular machine called the mitotic spindle, which the cell uses to pull replicated chromosomes apart during division. There are numerous compounds that affect spindle function, including several FDA-approved cancer drugs. These agents, which are called anti-mitotics, have a typical pattern of toxicity associated with their use. DZ-2384 is an anti-mitotic that behaves differently. In the study, researchers implanted or grew tumors in hundreds of rodents as models for various types of human cancer. They found that in animals receiving DZ-2384, tumors shrank as much as or more than when a conventional anti-mitotic was used, but with much less toxicity at effective doses. Animals receiving DZ-2384 also had markedly less peripheral neuropathy -- a type of debilitating nerve pain that often affects humans who take anti-mitotic drugs -- than those that received docetaxel, a conventional anti-mitotic. Peripheral neuropathy is one of the chief side effects of anti-mitotic chemotherapy, and it's often severe enough that doctors decide to stop administering chemotherapy, at least temporarily, in order to reduce patients' pain. But that's problematic as well, because once treatment is suspended, tumors tend to return, and often in forms that are resistant to drugs. Harran said understanding the unique way DZ-2384 functions in animals wouldn't have been possible without the cooperation of specialists from several fields over the past 15 years. "One piece of the puzzle after another came into place over time," Harran said. "Now, we have the chemistry, the biochemistry, the structural biology, the pharmacology and toxicology. We have a deep understanding of what this drug is doing." Harran believes the research could ultimately help large numbers of people with cancer, and he expects human clinical trials to begin within two years. "We have good reason to expect that human clinical trials of DZ-2384 will show that, at doses effective for treating a person's cancer, there will be much less risk of the peripheral neuropathy that can force clinicians to stop treatment," he said. Harran began his work with diazonamides as a fundamental chemistry research problem. In 1991, a study by other researchers described the toxin, which they had isolated from Diazona angulata. But when Harran and his team synthesized the chemical structure described in the published report, they discovered that it was not actually diazonamide. In 2001, Harran and colleagues published a study that corrected the chemical structure of diazonamide, and two years later they had synthesized the true structure in their lab. Next, they began studying what the molecule might be doing to block cells from dividing, and in 2007, they discovered that the synthetic diazonamides they had produced minimized undesirable toxic effects that are commonly associated with chemotherapy. Specifically, the synthetic forms of diazonamide had an unusually large "therapeutic window" -- the dosage range that would produce a desirable effect while minimizing undesirable side effects. In experiment after experiment, Harran said, the researchers found that synthetic diazonamides' therapeutic window was at least 10 times larger than that of traditional anti-mitotic drugs. In 2015, UCLA senior research associate Hui Ding prepared a special form of DZ-2384 that was engineered with a molecular-scale "tracking device" so scientists could monitor its activity and better understand how it worked. That helped colleagues at Montreal's McGill University -- Anne Roulston, Gordon Shore and Gary Brouhard -- confirm what they had come to suspect, and what others had looked for and failed to find earlier: that the compound binds to a protein called tubulin, which is a building block of mitotic spindles and a common target of anti-mitotic drugs. Armed with this information, researchers at Villigen, Switzerland's Laboratory of Biomolecular Research used a technique called X-ray crystallography to determine the structure of the DZ-2384 bound to tubulin. Their work offers a possible explanation for how DZ-2384 could disrupt dynamic tubulin polymers during cell division but spare those polymers in resting cells like neurons in the peripheral nervous system. And that, Harran said, is what appears to allow the compound to attack growing cancer cells while minimizing damage to healthy cells, which can lead to the painful side effects of conventional chemotherapy. "Patrick Harran is one of the most outstanding and creative synthetic organic chemists in the world," said Miguel García-Garibay, dean of the UCLA Division of Physical Sciences and professor of chemistry and biochemistry. "He bridges chemistry and biochemistry by using elegant synthetic chemical strategies to create and study compounds with important biological activity." Those who contributed to the research represent a wide range of expertise, from chemistry, biology and cancer research to neuroscience and pharmacology. Among them are Thomas Wilkie and Rolf Brekkenof UT Southwestern Medical Center in Dallas; and Michel Steinmetz of the Laboratory of Biomolecular Research. The research was partly funded by a sponsored research agreement between UCLA and Diazon Pharmaceuticals Inc., a Montreal-based company that owns the relevant intellectual property and was co-founded by Harran in 2013.

Macdonald P.R.,University of Manchester | Lustig A.,University of Basel | Steinmetz M.O.,Biomolecular Research | Kammerer R.A.,University of Manchester
Journal of Structural Biology | Year: 2010

Laminins are large heterotrimeric, multidomain proteins that play a central role in organising and establishing all basement membranes. Despite a total of 45 potential heterotrimeric chain combinations formed through the coiled-coil domain of the 11 identified laminin chains (α1-5, β1-3, γ1-3), to date only 15 different laminin isoforms have been reported. This observation raises the question whether laminin assembly is regulated by differential gene expression or specific chain recognition. To address this issue, we here perform a complete analysis of laminin chain assembly and specificity. Using biochemical and biophysical techniques, all possible heterotrimeric combinations from recombinant C-terminal coiled-coil fragments of all chains were analysed. Apart from laminin 323 (α3, β2, γ3), for which no biochemical evidence of its existence in vivo is available, these experiments confirmed all other known laminin isoforms and identified two novel potential chain combinations, laminins 312 (α3, β1, γ2) and 422 (α4, β2, γ4). Our findings contribute to the understanding of basement membrane structure, function and diversity. © 2010 Elsevier Inc.

Lopus M.,University of California at Santa Barbara | Manatschal C.,Biomolecular Research | Buey R.M.,Biomolecular Research | Bjelic S.,Biomolecular Research | And 4 more authors.
Biochemistry | Year: 2012

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ 32P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound 32P i. Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap. © 2012 American Chemical Society.

van der Vaart B.,Erasmus Medical Center | van Riel W.E.,University Utrecht | Doodhi H.,University Utrecht | Kevenaar J.T.,University Utrecht | And 20 more authors.
Developmental Cell | Year: 2013

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. Invitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/β1 and the components of the LL5β-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis. © 2013 Elsevier Inc.

Akhmanova A.,University Utrecht | Steinmetz M.O.,Biomolecular Research
Current Biology | Year: 2011

EB proteins accumulate at the tips of growing microtubules and recruit to them a multitude of factors to regulate microtubule functions. A new study suggests that EBs recognize microtubule ends by distinguishing between different states of the tubulin-bound guanine nucleotide. © 2011 Elsevier Ltd.

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